Simultaneous steroid oxygenation and 1-dehydrogenation with bacillus cereus

ABSTRACT

The invention disclosed herein relates to the process in which an 11-desoxy-3-oxygenated - Delta 4-steroid is subjected to the action of Bacillus cereus microorganisms thereby effecting simultaneous oxygenation (i.e., 11-hydroxylation) and Delta 1dehydrogenation of the steroid to produce an 11 Beta -hydroxy-3oxygenated- Delta 1,4steroid. For example, the 11-desoxy-3oxygenated- Delta 4steroid, Compound S, is converted directly by this microbiological oxygenation-dehydrogenation procedure to its 11 Beta -hydroxy- Delta 1-dehydro analog, prednisolone, which is known to be useful as an anti-inflammatory agent.

United States Patent Stoudt et al.

Apr. 2, 1974 SIMULTANEOUS STEROID OXYGENATION AND l-DEHYDROGENATION WITHBACILLUS CEREUS Inventors: Thomas H. Stoudt, Westfield;

Robert A. Long, East Brunswick, both of NJ.

Assignee: Merck & Co., Inc., Rahway, NJ. Filed: Dec. 18, 1970 Appl. No.:99,682

Related U.S. Application Data Continuation-in-part of Ser. No. 722,131,April 17, 1968, abandoned, which is a continuation of Ser. No. 601,313,Dec. 13, 1966, which is a continuation of Ser. No. 545,876, April 28,1966, abandoned, which is a continuation of Ser. No. 387,219, Aug. 3,1964, abandoned, which is a continuation-in-part of Ser. No. 208,608,July 9, 1962, abandoned.

U.S. Cl. 195/51 A, 195/51 E Int. Cl. C07c 167/08, C07c 167/14 Field ofSearch 195/51 E, 51 A References Cited OTHER PUBLICATIONS McAleer etal., Arch. Biochem. Bioanys. Vol. 73 pages 127-130 (1958).

Shirasaka et al., J. Gen Appl. Microbiol. (Japan) Vol. 7 supp. 1 1961pages 341-352.

Primary Examiner-Alvin E. Tanenholtz [57] ABSTRACT The inventiondisclosed herein relates to the process in which an 1l-desoxy-3-oxygenated -A-ster0id is subjected to the action of Bacilluscereus microorganisms thereby effecting simultaneous oxygenation (i.e.,11- hydroxylation) and A -dehydrogenation of the steroid to produce anl1B-hydroxy-3-oxygenated-A- steroid. For example, the11-desoxy-3-oxygenated-A steroid, Compound S, is converted directly bythis microbiological oxygenation-dehydrogenation procedure to its 11fi-hydroxy-A -dehydro analog, prednisolone, which is known to be usefulas an anti-inflammatory agent.

4 Claims, No Drawings SIMULTANEOUS STEROID OXYGENATION ANDl-DEHYDROGENATION WITH BACILLUS CEREUS This is a continuation-in-part ofSer. No. 722,131,

filed Apr. 17, 1968, now abandoned, which is a continuation of Ser. No.601,313, filed Dec. 13, 1966, which is a continuation of Ser. No.545,876, filed Apr. 28, 1966, now abandoned, which is a continuation ofSer. No. 387,219, filed Aug. 3, 1964, now abandoned, which, in turn, isa continuation-in-part of Ser. No. 208,608, filed July 9, 1962, nowabandoned.

- This invention relates to a novel method of making steroids, and moreparticularly, is concerned with the process of preparingllB-hydroxy-3-oxygenated-l,4- pregnadienes and llfi-hydroxy-3-oxygenated-1,4- androstadienes by subjecting3-oxygenated-4- pregnenes and 3-oxygenated-4-androstenes to the actionof a strain of Bacillus cereus.

The discovery of the remarkable therapeutic properties of prednisoloneand similar related compounds having a double bond at C-1(2) and ahydroxy substituent at C-ll has stimulated wide interest in findingsimple and more economical methods of preparing such compounds. Two ofthe principal difficulties encountered in the synthesis of prednisoloneand its related compounds from commercially available starting materialsare the introduction of the A -bond and the introduction of thell-hydroxy substituent. Although various methods have been developed forthe synthesis of these steroids, such processes are not entirelysatisfactory and other methods more suitable for the preparation ofthese steroids in high yields have been sought.

Methods for effecting the oxygenation of steroids by the action ofmicroorganisms are known in the art as, for example, microorganismsbelonging to the Order Mucorales introduce oxygen in the 6, 11, or 14positions of the steroid ring structure. Various methods are also knownfor effecting a dehydrogenation of steroids at the 1-position as, forexample, the conversion of hydrocortisone to prednisolone by Bacillussphaericus as described'by Stoudt, et al, in the Arch. Biochem &Biophys. 59, 30 (1955).

What is described herein, however, is a microbial method of producing anll-oxygenation and a 1- dehydrogenation of steroids within a singleprocess step. In accordance with the invention, Bacillus cereus is shownto effect the transformation of 3-oxygenated- 4-pregnenes or3-oxygenated-4-androstenes, including substituted derivatives thereof,to the corresponding 1 IB-hydroxy-S-oxygenated-1,4-pregnadienes or 113-hydroxy-3-oxygenated-l,4-androstadienes. In a preferred form of thepresent invention 4-pregnene-l7a, 2l-diol-3,20-dione (also known asReichsteins Compound S or ll-desoxy-l7a-hydroxycorticosterone) isconverted to prednisolone by the action of enzymes produced by theBacillus cereus microorganism.

Accordingly, it is an object of the present invention to provide amicrobial process for effecting lloxygenation and l-dehydrogenation ofsteroids in a single step. Other objectives will be apparent from thedetailed description of the invention hereinafter provided.

In accordance with the present invention, it is now found thatoxygenation-dehydrogenation of steroids is conveniently effected bysubjecting steroids to the action of any llB-hydroxylating-A-dehydrogenating strain of Bacillus cereus or to theoxygenatingdehydrogenating enzymes produced by such microorganismstrains.

The practice of this invention is particularly suitable, therefore, inconverting 1,2-dihydro-l l-desoxysteroids unsaturated at the4,5-position, i.e., steroidscontaining at least one hydrogen atom at C Cand C B to the corresponding A-1lB-hydroxy-steroidsin high yields. Thus,the invention provides a valuable means for producing prednisolone andcompounds related thereto.

The microorganism Bacillus cereus, employed herein, is identified andcharacterized in Bergeys Manual of Determinative Bacteriology (SeventhEdition), pages 617-8. Strains of Bacillus cereus capable of effectingthe desired conversion of steroids, as described herein, can be obtainedfrom known culture collections. For example, one preferred strain of aculture of Bacillus cereus, ATCC No. 14,737, is available to the publicand can be obtained from the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. Other preferred 1 1,B-hydroxylating-A-dehydrogenating strains of Bacillus cereus may be selected by thefollowing test method: a nutrient medium containing 1 g. of meatextract, 1 g. of glycerol, 0.1 g. of KH PO 0.15 g. of calcium chloride,and sufficient distilled water to make ml., is adjusted to pH 7.0,sterilized and inoculated with a culture of the particular strain ofBacillus cereus to be tested for its oxygenatingdehydrogenatingactivity. The inoculated nutrient medium is incubated at a temperatureof about 28C., while being agitated in the presence of oxygen, for aperiod of about 24-28 hours. To the resulting culture is then added asolution containing 25 mg. (0.025%) of Compound .S (4-pregnene-l7a,2l-diol-3,20-dione) dissolved in 0.25 ml. of dimethylformamide. Theculture containing the steroid compound is incubated, with agitation andaeration, for an additional period of about 16 hours at 28C. Thefermentation broth is adjusted to a pH of about 4.0, and extracted withlive 50 ml.-portions of ethyl acetate, and the ethyl acetate extractsare combined and evaporated to dryness in vacuo. A portion of theresidual material is dissolved in acetone and spotted on a paperchromagram which is developed using formamide as the stationary phaseand chloroform as the mobile phase; the presence of a band correspondingto the l lB-hydroxy-A -dehydro derivative of Compound S(l,4pregnadiene-l 16, 17a, 2l-triol-3,20-dione, i.e., prednisolone), andhaving an ultraviolet maximum of 242 mp. demonstrate that the teststrain is an llB-oxygenating-A -dehydrogenating strain of Bacilluscereus.

In carrying out the process of the invention the steroid to beoxygenated and dehydrogenated is subjected to the action ofoxygenating-dehydrogenating enzymes produced by growing a suitablestrain of Bacillus cereus. This is most conveniently accomplished bygrowing the microorganism under aerobic conditions in a suitablenutrient medium in intimate contact with the steroid to beoxygenated-dehydrogenated; the fermentation or growing of themicroorganism being continued until the desiredoxygenanon-dehydrogenation has occurred. Thus, the steroid to be soconverted can be incorporated directly in a suitable medium which isthen inoculated with an oxygenating-dehydrogenating strain of Bacilluscereus and incubated under aerobic conditions thereby effecting thedesired oxygenationdehydrogenation. Generally, the process is preferablyeffected by first growing the microorganism in a suitable fermentationmedium, then adding the steroid and continuing the cultivation of themicroorganism under aerobic conditions for sufficient time to effect thedesired transformations.

The steroid can be added to the nutrient medium as a suspension in asuitable solvent such as water, as a solution in a solvent such asacetone or propylene glycol, or in a finely-divided form, such as asolid micronized powder. In general, it is desirable that the steroid bepresent in very finely divided form in order to permit maximum contactwith the culture medium and insure completion of the reaction.

The process of the present invention can be effected in both stationaryand submerged cultures of Bacillus cereus growing under aerobicconditions, although for practical purposes it is most convenientlycarried out by growing the microorganism under submerged conditions in asuitable aqueous fermentation medium containing the steroid. The amountof the steroid which can be conveniently converted by our method willdepend in part upon the particular environmental conditions employed andthe specific steroid to be converted.

Aqueous nutrient mediums suitable for the growing of suitable strains ofBacillus cereus must contain sources of assimilable carbon and nitrogenas well as minor amounts of inorganic salts. Any of the usual sources ofassimilable carbon such as glucose, invert molasses, sucrose, and thelike, employed in fermentation media, can be used in carrying out theprocess of our invention. Similarly, complex sources of nitrogen usuallyemployed in commercial fermentation processes such as lactalbumin digest(Edamin") and corn steep liquor, or inorganic sources of nitrogen suchas sodium nitrate, ammonium nitrate, and the like, are satisfactory foruse in fermentation media. Minor amounts of inorganic salts, such assuitable soluble salts of magnesium, potassium, sodium and iron, areusually available in complex sources of carbon and nitrogen or may beconveniently added to the fermentation medium in minor amounts topromote maximum growth of the oxygenating-dehydrogenating microorganism.

The following is an example of a suitable aqueous nutrient medium whichcan be used in the process of the invention, although others may be usedas well.

Meat Extract 1% Glycerol 1% KH,PO. 0.1% CaCl, 0.15% pH 7.0

The addition of minor amounts of anti-foaming agents, although notessential, is desirable with some fermentation media in order to obtainmaximum yields of the desired converted product. The addition to certainfermentation media of a substituted oxazaline which is a non-volatile,amine-type, cationic surface active agent is particularly effective inreducing the amount of foam, although other anti-foam agents known to beuseful for this purpose can also be used.

As indicated above, the process of the invention is particularly usefulin the oxygenation-dehydrogenation of 3-oxygenated-4-pregnenes,3-oxygenated-4- androstenes, 3-oxygenated-4-estrenes, and derivativesthereof to obtain the corresponding ll-oxygenated-ldehydrogenatedsteroids. Thus, the process is applicable in general to saturated andunsaturated cyclopentanopolyhydrophenanthrene compounds having at leasta single hydrogen atom at C C and C B Suchcyclopentanopolyhydrophenanthrene compounds may be unsubstituted or maycontain substituents such as keto, hydroxyl, acyloxy, halide, alkyl andthe like at various positions of the cyclopentanopolyhydrophenanthrenenucleus. In addition, such compounds may have at the 17 position a ketolside chain, a saturated or unsaturated hydrocarbon side chain, acarboxy-containing side chain, and the like. Examples of classes of suchcyclopentanopolyhydrophenanthrene compounds that might be mentioned arepregnenes, androstenes, bile acids, sterols, hormones, sapogenins, andderivatives thereof. Thus, representative steroids having at least onehydrogen atom at C C and C such as progesterone, 4-pregnene-l7a-ol-3,20-dione, desoxycorticosterone, 4-pregnene-1 701,2 1 -diol- 3,20-dione,5-pregnene-3a-ol-20-one, 5,6-dichloro-4- pregnene-3B-ol-20-one, 5- or4-cholenic acid, diosgenin, l6B-methyl Compound S, l6a-methyl CompoundS, 6a, l6a-dimethyl Compound S, lo-methylene Compound S, 2-methylCompound S, 6a-methyl Compound S, l6a-hydroxy Compound S,'6a-fiuoroCompound S, and the like, can be converted at position one, two, andeleven, to obtain the corresponding A-l l-hydroxy derivatives.

For example, Compound S(4-pregnene-l70:,2l-diol- 3,20 dione) can beconverted to prednisolone with the following procedure: A sterileculture medium, such as those shown above, is first inoculated byintroducing a small amount of a slant containing anoxygenatingdehydrogenating strain of Bacillus cereus. The inoculatednutrient medium is incubated at a temperature of about 28C., while beingagitated in the presence of 0xygen for a period of about 24-28 hours. Atthis point, a solution of compound S is added to the fermentation mediumand the agitation and aeration is continued for about 16 hours, or untilthe oxygenationdehydrogenation is completed.

When the oxygenation-dehydrogenation is complete, theoxygenated-dehydrogenated steroid may be recovered from the fermentationbroth by extraction with a suitable water-immiscible organic solvent forthe oxygenated-dehydrogenated steroid. Suitable solvents for thispurpose that might be mentioned are chloroform, methylene chloride,organic acid esters, aromatic hydrocarbons, and the like. The solventsolution containing the desired oxygenated steroid then can beevaporated to yield the desired product which may be further purified byrecrystallization or other procedures conventional in the art.

Alternatively, the process of the invention can be effected bycontacting the enzymes produced by the fermentation of Bacillus cereuswith the steroid. This can be accomplished by recovering the enzymesfrom a fermentation broth in accordance with procedures known in theart, and intimately contacting such enzymes with a steroid in an aqueousmedium.

The following examples illustrate methods of carrying the process of thepresent invention, but it is to be understood that these examples aregiven for purposes of illustration and not of limitation.

EXAMPLE I Approximately 40 m1. of the nutrient medium shown hereinabovein 250 ml. Erlenmyer shake-flasks is sterilized for minutes at 121C.After sterilization, the medium is inoculated with a loop of growth froma slant of a strain of Bacillus cereus culture, ATCC No. 14,737. Themixture is then agitated on a rotary shaker at 220 rpm using a 2 inchstroke and maintaining the temperature at 28C. for a period ofapproximately 24 hours. At the end of the 24-hour period, the growth isused to inoculate l0 flasks of the same medium using a 10% inoculumlevel.

At the end of a 24 hour period, 1 ml. of ethanol containing 10 mg. ofCompound S is charged to each flask and agitation of the medium iscontinued for a period of 16 hours.

Following the conversion period the steroid containing fermented mediumis adjusted to a pH of 4.0 and extracted five times with one-half volumeamounts of ethyl acetate. Crystalline prednisolone then is isolated fromthe combined extractsby removal of ethyl acetate in vacuo.

EXAMPLE II Following the process described in detail above and using themedium given below in place of the one used in Example I there isproduced a similar conversion of Compound S to prednisolone.

Meat Extract 0.2% Polypeptone 0.2% Glycerol 0.2% pH I 7.0

EXAMPLE III Following the procedure described in Example I and using1,5,25,50 mg. of Compound S in place of 10 mg. of Compound S thecompound prednisolone is produced.

EXAMPLE IV Following the process eescribed in Example II, 16amethylcompound S is substituted for Compound S and 16a-methy1prednisolone isproduced.

EXAMPLE V 6a-fluoroprogesterone;

6-ch1oro- 1 7a-acetoxy-A -progesterone;

6B-hydroxy- 16a, 1 7a-epoxyprogesterone;

6,8-fluorol 7a-hydroxyl 9-norprogesterone; and

1 9-norprogesterone.

Many of the compounds which are prepared in accordance with the processof this invention are themselves therapeutically useful because ofhormonal activity. Others are especially useful as intermediates for thepreparation of therapeutically useful steroids. Still others, althoughexhibiting an appreciable degree of physiological activity, areespecially useful as intermediates for the preparation of derivativeshaving increased physiological activity. These conversions may beeffected by procedures well known in the art. For example, the ll-hydroxylated steroids are converted to the corresponding 9a-fluoro-llfl-hydroxy steroids by the well known procedure of Fried and Sabo;l7-oxoandrostadienes are readily converted to the correspondingl7B-hydroxy-l7a ethynyl or haloethynyl derivatives by reaction withorganometallic compounds; and the like.

While the invention has been described with particular reference tocertain preferred embodiments thereof, it will be understood thatvarious additions and modifications may be madewhich are within theskill of the art.

What is claimed is:

1. The process which comprises subjecting a steroid substrate comprisinga N-steroid unsubstituted at C-l 1 to the oxygenating-dehydrogenatingaction of an 1 1B- hydroxylating-A -dehydrogenating strain of Bacilluscereus, thereby producing the corresponding 1 1,3- hydroxy-A -steroid,and recovering the llB-hydroxy- A"-steroid thus produced from theoxygenateddehydrogenation mixture; said lIB-hydroxylating-N-dehydrogenating strain being characterized in that, when an established28-hour culture of said strain containing approximately 0.025% of4-pregnene-17a,21- dio1-3,20-dione is incubated for about 16 hours atapproximately 28C., and the ethyl acetate-extractible components of theresulting fermentation broth are subjected to paper chromatography usingformamidechloroform system, there is obtained a band corresponding to1,4-pregnadiene-l 1B, l7a,21-triol-3,20-dione and having ultravioletabsorption maximum of 242 u.

2. The process as defined in claim 1 in which the steroid substrate isselected from the group which consists of 1,2-dihydro-11-desoxy-3-oxygenated-4-pregnenes; l ,2-dihydro-11-des0xy-3-oxygenated-4-androstenes; and 1,2-dihydro-l1-desoxy-3-oxygenated-4-estrenes, and the 1 lB-hydroxy-A -steroidproduct is the corresponding l 1,8-hydroxy-3-oxygenatedl ,4-pregnadiene;l 1 B-hydroxy-3-oxygenatedl ,4-androstadiene; or 1 1,8-hydroxy-3-oxygenated 1,4-estradiene.

3. The process as defined in claim 1 in which the 1 1B-hydroxylating-Adehydrogenating strain of Bacillus cereus utilized isthat having the ATCC No. 14,737.

4. The process as defined in claim 1 in which prednisolone is producedby subjecting 4-pregnene-17a, 21-diol-3,20-dione to the action of lIB-hydroxylating- A'-dehydrogenating enzymes produced by Bacilluscereus, ATCC No. 14,737, microorganisms.

2. The process as defined in claim 1 in which the steroid substrate isselected from the group which consists of1,2-dihydro-11-desoxy-3-oxygenated-4-pregnenes;1,2-dihydro-11-desoxy-3-oxygenated-4-androstenes; and1,2-dihydro-11-desoxy-3-oxygenated-4-estrenes, and the 11 Beta -hydroxy-Delta 1,4-steroid product is the corresponding 11 Beta-hydroxy-3-oxygenated-1,4-pregnadiene; 11 Beta-hydroxy-3-oxygenated-1,4-androstadiene; or 11 Beta-hydroxy-3-oxygenated 1,4-estradiene.
 3. The process as defined in claim1 in which the 11 Beta -hydroxylating- Delta 1-dehydrogenating strain ofBacillus cereus utilized is that having the ATCC No. 14,737.
 4. Theprocess as defined in claim 1 in which prednisolone is produced bysubjecting 4-pregnene-17 Alpha , 21-diol-3,20-dione to the action of 11Beta -hydroxylating- Delta 1-dehydrogenating enzymes produced byBacillus cereus, ATCC No. 14,737, microorganisms.